Diseases caused by retroviruses such as AIDS and leukemia have intensified the need to understand the mechanisms of retrovirus replication. Our primary objectives are to understand how reverse transcription of viral mRNA occurs and how the cDNA products are integrated into the genome of infected cells. Owing to their similarity to retroviruses, LTR-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. During the synthesis of cDNA, reverse transcriptase (RT) generates a series of highly specific intermediates. We identified residues of Tf1 RT that recognize specific intermediates of cDNA by screening large numbers of RT mutants. A combination of genetic assays and physical analyses identified a set of 35 mutations that inhibited integration without reducing reverse transcription. Our experiments focused on a cluster of mutations in ribonuclease H (RNase H) that included a region with five single amino acid substitutions in a five?amino acid segment. Surprisingly, these mutations in RNase H did not reduce the levels of full-length double stranded cDNA. Crystallographic studies by Dr. Edward Arnold and colleagues indicated that the corresponding residues of HIV-1 RT interact directly with the PPT. This observation led us to test whether the mutations in the RNase H of Tf1 were defective for the cleavages on either side of the PPT that generate the primer, or the cleavage that removes the PPT after it has primed plus strand synthesis. Defects in the position these cleavages would alter the sequences at the 3' end of the minus strand and as a result have a drastic impact on the ability of IN to catalyze strand transfer. The sequence from the 3? ends of the minus strand cDNA from Tf1 particles was determined using ligation mediated PCR. The mutations clearly increased the levels of cDNA that retained the PPT at the 3? end. In order to determine whether this added sequence resulted from a defect in the cleavages that generate the PPT or the cleavage that removes the PPT after synthesis of plus strand DNA, the 5? end of the plus strand DNA was analyzed. The results of primer extension provided strong evidence that the mutations in RNase H specifically inhibited the removal of the PPT RNA from the 5? end of the plus strand. As a result, our data identified a cluster of conserved amino acids in RNase H that have the specific function of removing the PPT. [unreadable] [unreadable] In addition to removing the PPT primer from the plus strand, RNase H is known to remove the primer from the 5? end of the minus strand DNA. Tf1 uses a unique mechanism of self-priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus strand synthesis with an 11 nucleotide RNA removed from the 5? end of its own transcript. We tested whether the self-primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self-primer. This indicates that integration of the Tf1 cDNA relies on mechanisms other than reverse transcription to remove the primer. [unreadable] [unreadable] Our analysis of the genome sequence of S. pombe revealed a strong clustering of pre-existing LTRs associated with the 5? end of ORFs. Experiments based on the production of new integration events revealed that the association of Tf1 with LTRs was the result of integration preference. To define the determinants of the target sites we developed an in vivo assay for integration using a plasmid that contained ade6 as the target and a plasmid with Tf1 that induced transposition. The version of Tf1 we expressed contained a neo gene to cause target plasmids with insertions to gain resistance to kanamycin. When Tf1-neo was expressed, the plasmid with ade6 served as an efficient target for integration. We isolated 50 separate insertions in the intact target plasmid and found ninety-five percent occurred within a 160 nt region in the ade6 promoter. To determine which sequences of Ade6 were required for efficient integration, we created a series of 10 deletions within the target plasmid. This analysis revealed that the 160 nt region of the promoter was the only sequence that was required for efficient integration. We asked whether promoter activity was required for integration by measuring transcript levels of ade6. Deletions of sequence on either side of the 160 nt region caused five to ten-fold reductions in ade6 mRNA. Nevertheless, the deletions caused no reduction in integration efficiency. These results indicated that transcription was not important for target site activity. We next considered whether transcription factors themselves were directing the integration of Tf1. To identify positions where factors bind in the promoter of Ade6, we used micrococcal nuclease mapping. We observed a strong correlation between micrococcal sensitive sites and the position of the prominent insertion sites. This suggested transcription factors played a role in directing Tf1 integration. Hoffman and colleagues showed previously that the transcription factor Atf1p binds to and activates the promoter of fbp1. We tested whether the promoter of fbp1 is a target of Tf1 integration using the target plasmid assay. We found that the fbp1 promoter was a target for Tf1 insertion and that the majority of the insertions occurred 40 nt from the position where Atf1p binds. A mutation that blocks the binding of Aft1p caused a significant reduction in Tf1 integration at the promoter of fbp1. These data indicate that Atf1p is responsible for targeting Tf1 to specific insertion sites in the fbp1 promoter.